Labeled nucleotide analogs

ABSTRACT

Labeled reactant compositions, and particularly labeled nucleic acid reaction compositions that include structural components including double-stranded nucleic acids. In some embodiments, the structural components maintain potentially damaging labeling components sufficiently distal from the reactant portion of the molecule such that damaging effects of the label group on other reaction components, such as enzymes, are reduced, minimized and/or eliminated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/688,667 filed Nov. 29, 2012, which is acontinuation of Ser. No. 13/115,288 filed May 25, 2011, which is acontinuation application of U.S. patent application Ser. No. 12/403,090filed Mar. 12, 2009, which claims the benefit of U.S. Provisional PatentApplication No. 61/069,247 filed Mar. 13, 2008, the full disclosures ofwhich are hereby incorporated herein by reference in their entirety forall purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

In the analysis of biological processes, researchers are constantlylooking for new and better ways to eavesdrop on both the individualreactions that make up complex biological systems, as well as observethe operation of those systems as a whole. In doing so, researchers havedeveloped methods, systems and compositions that employ artificiallylabeled molecules as model constituents for those reactions and systems.Observation of the model molecules is rendered facile by the presence ofthe labeling group. Such labels include radioactive compounds orradiolabels, chromophoric labels that absorb and/or reflect light ofdifferent wavelengths to provide colored indications of an event,chemiluminescent labels that can spontaneously emit light in response toa particular chemical event, fluorescent labels that emit light inresponse to excitation by light of a different wavelength, and reportersystem labels, that provide an exogenous, assayable activity or propertyto indicate the presence, absence or change in the model molecule. Suchreporter labels often include exogenous enzymes, binding molecules orthe like that are capable of being identified and even quantified.

In attaching label groups to different model reaction constituents, oneruns the risk that the presence of the label will adversely impact thereaction being observed. For example, large hydrophobic labeling groupscan present issues of steric interference with the progress of thereaction of interest by blocking or not properly interacting with theother reaction constituents. Likewise, labeling components that impactthe chemical properties of the model compound or the reactionenvironment can similarly adversely impact reaction conditions. In othercases, the properties of the label itself may adversely affect thereaction components. For example, the presence of fluorescent moleculesin close proximity to enzymatic reaction components can lead to decay inthe level of enzyme activity through photo-chemically induced reactionintermediates or other impacts.

Accordingly, it would be desirable to provide reaction components thatprovide remedies to some of the issues created by the incorporation oflabeling groups on reaction constituents. The present invention providesthese and other solutions.

BRIEF SUMMARY OF THE INVENTION

The invention generally relates to labeled compounds that compriselinker groups coupling the labeling moiety to the reactive portion ofthe compound such that the labeling group is maintained a sufficientdistance away from the reactive portion that potential negative impactsof the label moiety on the reactive portion or other compounds, enzymesor other reactants that react with the reactive portion, are avoided,reduced or otherwise mitigated. In one aspect, the invention provides alabeled reactant composition, that comprises a reactant component, alabel component, and a linker component coupling the label component tothe reactant component. The linker component maintains the labelcomponent at a functional distance from the reactant component of atleast 2 nm.

In another aspect, the invention provides a composition, comprising anenzyme, and a substrate for the enzyme, the substrate comprising areactant component, a label component and a linker component. The linkercomponent maintains the label component at a functional distance awayfrom the reactant component that a negative impact of the labelcomponent on the enzyme is reduced by at least 20%.

In a further aspect, the invention provides a method of monitoring anenzyme reaction. The method generally comprises providing a reactionmixture comprising the enzyme and at least a first reactant composition,the reactant composition comprising a compound having a reactantcomponent, a fluorescent label component, and a linker component joiningthe reactant component to the label component, wherein the linkercomponent maintains the label component at a functional distance awayfrom the reactant component that a negative impact of the labelcomponent on the enzyme is reduced by at least 20%. The reaction mixtureis then illuminated to excite the fluorescent label component, and afluorescent signal from the reaction mixture characteristic of theenzyme reaction is detected.

The invention also provides methods of monitoring nucleic acid synthesisreactions. The methods comprise contacting a polymerase/template/primercomplex with a fluorescently labeled nucleotide or nucleotide analoghaving a nucleotide or nucleotide analog component, a fluorescent labelcomponent, and a linker component joining the nucleotide or nucleotideanalog component to the label component, wherein the linker componentmaintains the label component at a functional distance away from thenucleotide or nucleotide analog component that a negative impact of thelabel component on the enzyme is reduced by at least 20%. Acharacteristic signal from the fluorescent dye is then detected that isindicative of incorporation of the nucleotide or nucleotide analog intoa primer extension reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates one exemplary aromatic linker for usein the present invention.

FIG. 2 schematically illustrates a synthesis for incorporating aromaticlinkers of the invention into nucleoside phosphate analogs comprising:coupling of aryl bromide (2) and aryl alkyne (1) in step (a); followedby phosphorylation of the resulting alcohol (3) in step (b); couplingthe phosphate of the resulting phosphorylated compound (4) to theterminal phosphate of a nucleotide by carbonyldiimidizole activation insteps (c)1 and (c)2; deprotecting the amine in step (c)3; then adding adye moiety using standard NHS chemistry in step (c)4 to yield the dyelabeled nucleoside hexaphosphate analog (5).

FIG. 3 schematically illustrates a synthesis for incorporating anoligoproline linker of the invention into a dye labeled nucleotideanalog comprising: coupling an NHS activated iodoacetamide (1) to anamino-linker nucleotide (2) in step (a) to yield the nucleotide analog(3); coupling this to the thiol group of the peptide Gly-(Pro)6-Cyspeptide (4) in step (b); then coupling a dye group is to the aminoterminus on the glycine residue as the TFP ester in step (c) to form thedye labeled nucleotide analog (5).

FIG. 4 schematically illustrates alternative double stranded nucleicacid linkers of the invention. In FIG. 4A the label component isprovided directly linked to the reactant portion. In FIG. 4B the labelcomponent is provided indirectly linked via the complementaryoligonucleotide. In FIG. 4C the label component is provide on a hairpinloop structure.

FIG. 5 illustrates improved photostability of polymerase complexes usinglinkers of the invention.

FIG. 6 schematically illustrates one embodiment of a system for use withthe compositions and in the methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention is directed to labeled reactants and their usesthat have improved characteristics for use in analytical operations. Inparticular, provided are compositions and methods of using suchcompositions, in which the label component of the molecule, while stillattached to the reactant component of the molecule, is nonethelessprovided sufficiently distal to that reactant component to minimizepotential adverse effects of that labeling component on the reaction ofinterest. Specifically, the linkage between the labeling group and thereactant group is configured to maintain sufficient distance between thetwo, such that negative or adverse impacts of the labeling group on thereaction of the reactant with other components is minimized. Ofparticular interest is the use of linker moieties that provide for themaintenance of fluorescent labeling groups outside of the active site ofthe enzyme(s) that are involved in the reaction of interest, andpreferably, sufficiently distant from key portions of enzymes that arereacting with the molecules to which such labels are attached, so thatany adverse or negative impacts of the labeling group on the enzyme orother reactants interacting with the enzyme, are reduced, minimized oreliminated.

For purposes of description, the reactant that bears the labeling groupwill be referred to herein as the first reactant, which is comprised ofa label portion and a reactant portion. The reactant portion denotes theportion of the first reactant that serves as the reactant in thereaction of interest, with or without the label group. For example, innucleic acid reactions utilizing fluorescently labeled nucleotideanalogs as the first reactant, the label portion that includes thefluorescent dye component is connected to a nucleotide or nucleotideanalog that forms the reactant portion.

Typically, the linker configurations of the present invention providefor a linkage of sufficient length and sufficient structure or rigidityto maintain the desired distance between the label portion and thereactant portion of the first reactant during a given reaction, suchthat adverse impacts of the label portion on either of the reactantportion or other reaction components which react with the reactantportion are reduced, minimized or eliminated. In particular, whileactual linker length is one important factor in the maintenance of thelabel at a desired distance, functional length, e.g., the actualdistance maintained between the label portion and the reactant portion,is believed to be the key influence, in that most adverse effects arebelieved to based upon relative proximity between the labels and theother reactants which suffer adverse impacts.

In a first aspect, the maintenance of sufficient distance between thelabel portion and the reactant portion of the first reactant may becharacterized as a function of the desired reduction in a given adverseimpact, as compared to similar molecules in which the label group iswithin a distance of about 1 nm of the reactant group. By way ofexample, in a nucleic acid polymerase mediated primer extension reactionthat uses labeled nucleotide analogs where the label group is coupled tothe nucleotide portion by a relatively short hexyl linker, e.g., alinkage that depending upon the level of coiling, can be from less than1 nm to about 2 nm in a fully stretched configuration, it has beenobserved that when the reaction is carried out under excitationillumination, that within the first minute of the reaction, thepolymerase activity can be depleted by as much as 50%, depending uponthe illumination conditions, reaction conditions, and fluorescentmaterials present.

As noted above, in this context, the compositions of the presentinvention typically provide sufficient distance between the labelcomponent and the reactant component as to reduce such photo-induceddecrease in enzymatic activity over that which occurs where thefluorescent label component is closer to the reactant component, i.e.,nucleotide, such as is the case where a hexyl linker is employed.

In accordance with the present invention, substitution of the linkagesdescribed herein, will yield a 20% reduction in the amount of thedepletion over the same time period as compared to a linkage that hasthe shorter functional length (e.g., less than 2 nm), preferably a 50%reduction in that depletion, more preferably at least a 90% or even atleast a 95% reduction in that depletion over the same time period. Withrespect to a 90% reduction, for example, if the normal depletion inactivity is 50% using a labeled molecule with a 1 to 2 nm linkage, thenthe corrected depletion would be no more than 5% (a 90% reduction in the50% depletion, meaning one would regain the 45% of activity that wouldotherwise be lost).

In another aspect, the reactants of the invention are characterized bythe specific distances provided between the reactant portion and thelabel portion. Because of differences in the relative flexibility ofdifferent linkages, such distances are generally stated in terms of anoperating or functional distances, e.g., the average maintained distancebetween label group and reactive group. In the case of linear linkages,such distances may be provided using polymers or other linear structuresthat have persistence lengths of at least the desired distances.Alternatively, some linkages may provide a spatial separation based uponthe volume of the linkage, e.g., PEG linkers that may exist as a randomcoil that provides a consistent spatial separation between the labelgroup and the reactive group.

While precise distances or separation may be varied for differentreaction systems to obtain optimal results, in many cases it will bedesirable to provide a linkage that maintains fluorescent label groupsat least 2 nm from the reactant portion of the first reactant, and insome cases at least 5 nm from the reactant portion of the first reactantor even at least 10 nm from the reactant portion.

A number of linkers may be employed that will provide the desiredseparation between label and reactant portion of the molecule. Forexample, alkyl linkers may be used that provide a useful distancebetween the reactant group and the dye group. For example, longeramino-alkyl linkers, e.g., amino-hexyl linkers, have been used toprovide dye attachment to nucleotide analogs, and are generallysufficiently rigid to maintain such distances.

In preferred aspects, however, providing linkers with desired functionallengths typically involves the use of more rigid chemical structures insuch linkers. Typically, such rigid structures include laterally rigidchemical groups, e.g., ring structures such as aromatic compounds,multiple chemical bonds between adjacent groups, e.g., double or triplebonds, in order to prevent rotation of groups relative to each other,and the consequent flexibility that imparts to the overall linker.Alternatively or additionally, secondary chemical structures may be usedto impart rigidity, including, for example helical structures, sheetstructures, and the like, as well as structures that employ cooperativemolecules in providing rigidity, e.g., complementary molecularstructures.

As noted, some linkers according to the invention derive rigiditythrough the internal chemical structure of the linker molecules. Forexample, linker molecules may derive their rigidity through a reductionin the number of single bonds that could yield points of rotation, andthus, flexibility in the linker. As such, the linkers will typicallycomprise double bonds, triple bonds or ring structures, which willprovide the increased rigidity. Examples of double and/or triple bondedlinker structures include, for example, conjugated alkynes, conjugatedalkenes, aryl alkynes, and the like. While illustrated as polymericstructures of repeating monomeric subunits, it will be appreciated thatthe linkers of the invention may comprise mixed polymers of differingmonomeric subunits.

Linkers that comprise ring or aromatic structures can include, forexample aryl alkynes and aryl amides. One exemplary aromatic linker isillustrated in FIG. 1, which shows an aryl alkyne, linking a dye groupto a nucleotide or nucleotide analog. Although illustrated as a dimer,it will be appreciated that the length of the linker is readilyincreased by the addition of monomers in the synthesis process that isschematically illustrated in FIG. 2. As shown, Heck coupling of the arylbromide (2) and aryl alkyne (1) in step (a) is followed byphosphorylation of the resulting alcohol (3) in step (b). The phosphateof the resulting phosphorylated compound (4) is coupled to the terminalphosphate of a nucleotide by carbonyldiimidizole activation in steps(c)1 and (c)2, followed by deprotection of the amine in step (c)3. Thedye moiety is then added using standard NHS chemistry in step (e)4 toyield the dye labeled nucleoside hexaphosphate analog (5).

Other examples of the linkers of the invention include oligopeptidelinkers, and in particular, oligoproline linkers that also include ringstructures within their structure. Oligoproline linkers will typicallyhave the structure shown in FIG. 3. An exemplary strategy for linkingdye groups to nucleotides or nucleotide analogs is also illustrated inFIG. 3. In particular, a NHS activated iodoacetamide (1) is coupled toan amino-linker nucleotide (2) in step (a) to yield the nucleotideanalog (3). This is coupled to the thiol group of the peptideGly-(Pro)₆-Cys peptide (4) in step (b). The dye group is then coupled toamino terminus on the glycine residue as the TFP ester in step (c) toform the dye labeled nucleotide analog (5).

The linkers used in the context of the invention may additionally oralternatively derive rigidity from secondary, tertiary or evenquaternary structures. For example, in some cases, polypeptide linkersmay be employed that have helical or other rigid structures. Suchpolypeptides may be comprised of rigid monomers, e.g., as in theoligoproline linkers described previously, which derive rigidity bothfrom their primary structure, as well as from their helical secondarystructures, or may be comprised of other amino acids or amino acidcombinations or sequences that impart rigid secondary or tertiarystructures, such as helices, fibrils, sheets, or the like. By way ofexample, polypeptide fragments of structured rigid proteins, such asfibrin, collagen, tubulin, and the like may be employed as rigid linkermolecules.

In a related aspect, double stranded nucleic acids can be used toprovide both the requisite length and rigidity as a linker. Similarly,related structures, such as double stranded peptide nucleic acids(PNAs), or DNA/PNA hybrid molecules may be employed as the linkers (See,e.g., FIG. 3, below). By way of illustration, the persistence length ofdouble stranded nucleic acids, i.e., the length up to which thestructure behaves more rod-like than rope-like, is approximately 50 nm,allowing for facile construction of rigid linkers up to and even beyondthis length.

II. Nucleotide Analogs and Polymerases

In particularly preferred aspects, the compounds of the inventioncomprise fluorescently labeled nucleotides or nucleotide analogs thatare used in enzymatic reactions, and particularly polymerizationreactions in which the fluorescent label is excited during the synthesisprocess. In particular, fluorescently labeled nucleotide analogs havebeen shown to negatively impact the activity of nucleic acidpolymerases, when the reaction between the polymerase and the analog iscarried out under conditions that excite the fluorescent label, i.e.,under excitation illumination. In particular, a fluorescent label groupor fluorophore, excited by exposure to electromagnetic radiation at anexcitation wavelength can transition into a triplet state. Subsequentrelaxation of the triplet state fluorophore can then lead to generationof reactive oxygen species. Without being bound to a particular theoryof operation, it is believed that generation of these reactive specieswithin or sufficiently proximal to the active site of enzymes such aspolymerases, can lead to damage to one or both of the fluorophore and/orthe enzyme processing the fluorescently labeled reactant.

Previous approaches have sought to mitigate the impacts of these speciesby including agents within the overall reaction that mitigate theproblematic issues, e.g., oxygen scavenging agents (See, e.g., PublishedU.S. Patent Application No. 2007-0161017, which is incorporated hereinby reference in its entirety for all purposes). In contrast, the presentinvention, instead of or in addition to providing mitigating agentswithin the reaction mixture, provides the fluorescent labeling groupaway from key components of the enzyme, such as the active site. Asnoted above, this is accomplished by providing the compounds of theinvention with a linker molecule between the nucleotide or nucleotideanalog and the label group, which linker group is sufficiently long andpossessing of sufficient structure to maintain the label in a positionaway from the enzyme, or key portions thereof. As such, the linkermolecules will typically be longer than a threshold distance and will besufficiently rigid to maintain the fluorophore at a sufficient distanceduring a reaction, as set forth above. In the context of polymerasemediated synthesis reactions using excited fluorescently labelednucleotides or analogs thereof, it will be appreciated that thesufficient distance may be characterized as set forth above, e.g., basedupon a prescribed reduction in the level of depletion of polymeraseactivity as compared to nucleotides where the fluorescent group iswithin less than 2 nm.

The nucleotide analogs of the invention may comprise any of thebiologically relevant nucleotides or deoxynucleotides or analogs thereofincluding ribonuclotides and deoxyribonucleotides or analogs thereof.Typically, such nucleotides or analogs include, adenosine, thymidine,guanosine, cytidine, uracil, and analogs of these. The analogs maycomprise nucleoside triphosphates, or in preferred aspects may includeadditional phosphate groups, e.g., tetraphosphates, pentaphosphates,hexaphosphates, heptaphosphates, or greater. Examples of some of theseanalogs are described, for example, in Published U.S. Patent ApplicationNos. 2003-0124576 and 2007-0072196, as well as U.S. Pat. Nos. 7,223,541and 7,052,839, the full disclosures of which are incorporated herein byreference in their entirety for al purposes.

The compounds of the invention will typically include any of a varietyof fluorophores as the labeling portion, including, for example,fluorescein based dyes, rhodamine based dyes, cyanine based dyes (Cy3,Cy5, and others, available from GE Healthcare). Other fluorophores thatare readily commercially available include those available fromInvitrogenlMolecular Probes (Carlsbad, Calif.), such as the Alexa® dyes,e.g., Alexa 488, 555, 568 and 660.

As noted previously, the linkage between the labeling portion and thereactant portion of the first reactant, e.g., a fluorescent dye and anucleotide analog, is configured to provide sufficient linker length andstructure as to maintain the label a sufficient distance from thereactant portion such that negative impacts of the label portion on thereactant portion, or those reaction components that interact with thereactant portion, are minimized or avoided. In the context of nucleicacid sequencing that employs real-time detection of the interaction oflabeled nucleotides with polymerase enzymes, one impact that is soughtto be avoided, is the impact of the excited fluorophore, or itsby-products, on the activity of a polymerase enzyme interacting with thenucleotide. In particular, and as noted above, it has been found thatexcitation of fluorescently labeled nucleotides as they interact withnucleic acid polymerase molecules, can yield a substantial reduction inthe activity of those polymerase molecules over time (See, PublishedU.S. Patent Application No. 2007-0161017, which is incorporated hereinby reference in its entirety for all purposes). Again, without beingbound to a particular theory of operation, it is believed thatby-products of the fluorescent excitation reaction cause damage to theportions of the polymerase that are proximal to the fluorophore, e.g.,the active site.

As noted previously, the linkers of the invention serve to distance thefluorophore from the reactant portion, such that the negative impact isreduced or avoided. As noted previously, the reduction in the negativeimpact will be at least 10%, more preferably, at least 20%, and stillmore preferably, at least a 50% reduction in the negative impact, ascompared to linkers that maintain the fluorophore within 2 nm of thereactant portion of the molecule. In the context of a reduction inphotodamaging effects, therefor, it will be appreciated that the analogsof the invention, e.g., including the linkers described herein, willresult in a decrease in the activity reduction of at least 10%, morepreferably 20% and still more preferably, at least 50%, as compared tothe reduction in activity when using a similarly labeled nucleotideanalog having a linkage with a persistence length less than 2 nm.

While linker length is typically a function of providing a sufficientnumber of monomers or other linkage units in the linker molecule,providing sufficient structure typically involves adjusting the natureof those monomeric units so as to provide a structurally more rigidlinker.

As noted above, in certain preferred aspects, a nucleic acid linker thatcomprises a double stranded portion to impart rigidity is used as thelinker group between the reactant component, e.g., the nucleotide ornucleotide analog, and the label component, e.g., the fluorophore. Suchdouble stranded nucleic acids may comprise distinct but complementarynucleic acid strands that are hybridized together, where one or bothstrands bear a label component. Alternatively, the nucleic acid linkermay comprise a single molecule with complementary portions, such thatthe molecule self hybridizes to form a hairpin loop structure, where thelabel component is provided at a point on the loop, distal to thereactant portion. The use of nucleic acid linker structures providesadvantages of ease of synthesis of the labeled linker, usingconventional DNA synthesis and dye coupling techniques, and resultantcontrol of linker length, e.g., approximately 0.3 nm of distanceimparted for each added monomer in the linker portion. Consequently, onecan easily adjust the length of the linker to accommodate more or lesssensitive reaction systems. Additionally, the ability to adjust therigidity of the linker, in real time provides interesting reactioncontrol elements, e.g., by adjusting the integrity of the hairpinstructure by modifying the hybridization conditions for the linker,e.g., adjusting salt, temperature, or the like. These linkers are alsoreadily coupled to nucleotide analogs, whether coupled through groups onthe nucleobase, the ribosyl moiety, or through one of the phosphategroups (e.g., alpha, beta, gamma, or others in the case of tetra, penta,or hexa phosphate analogs, or others).

Labeled nucleotide analogs according to this aspect of the invention areschematically illustrated in FIGS. 4A, 4B and 4C. In particular, FIGS.4A and B illustrate a nucleic acid linker that employs two distinct butcomplementary nucleic acid strands 402 and 404 to impart rigidity andlength to the linker, so as to provide sufficient functional distancebetween the reactant component, e.g., nucleotide 406, and the labelcomponent, e.g., the fluorophore 408. As shown in FIG. 4A, the labelcomponent may be provided directly linked to the reactant portionthrough the same nucleic acid strand, e.g., oligonucleotide 402. Thisprovides an advantage of maintaining a linked label component regardlessof whether the reaction conditions are particularly suited for continuedhybridization of the two nucleic acid strands.

Alternatively, the label component may be provided indirectly linked tothe reactant portion, as shown in FIG. 4B via the complementaryoligonucleotide 404, in order to provide flexibility in coupling oflabel components to reactant components. In particular, one can assignthe particular fluorophore to a particular reactant portion by simplyintroducing a new labeled nucleic acid that hybridizes with the linkerportion of the reactant portion. Additionally, a hybridized orhybridizable label component provides additional flexibility incontrolling label association with the reactant that is not available inlinkers that involve covalent attachment of the label component to thereactant component. In particular, by adjusting the environmentalconditions, one can adjust the level of hybridization between the labelcomponent and the reactant component for any of a variety of uses.

In still another aspect, a single nucleic acid strand that includesinternally complementary portions, may be employed as the linker, suchthat the complementary portions can hybridize to form a hairpin loopstructure 410, as shown in FIG. 4C. As will be appreciated, coupling ofthe nucleotide to the oligonucleotide linker may be accomplished througha variety of known linkage chemistries. For example, as shown in FIG. 4,linkage group L may include a variety of groups, including, for examplealiphatic linker groups such as the alkyl or other groups previouslydescribed herein, such as hexyl linkers. Linkage bond X may include anyof a variety of linkages, including thioether linkages, peptide bonds,bifunctional groups, such as malemide-spacer-NHS linkers, to facilitatelinkage of the nucleotide to the oligonucleotide linker, and the like.In addition, and as indicated in FIG. 4, the nucleotide may comprise anyof a variety of nucleotides or nucleotide analogs, including, forexample, nucleoside polyphosphates that include three, four, five oreven more phosphates in the phosphate chain, e.g., where n=from 3 to 7.Likewise, the base groups may be similarly modified to achieve a desiredapplication, e.g., substitution of the natural bases with universalbases, or the like.

While preferred embodiments of the invention relate to nucleic acidmolecules, including nucleotides, nucleotide analogs, oligonucleotidesand polynucleotides, that include the foregoing features, the principlesof the invention are equally applicable to a broad range of reactants,labeling groups and their counterpart enzyme systems, such as kinases,phosphatases, and their substrates. For ease of discussion however, theinvention is described in terms of fluorescently labeled nucleotides ornucleotide analogs, and their interaction with nucleic acid processingenzymes such as polymerases, including reverse transcriptases,nucleases, ligases, and the like, with DNA polymerases being aparticularly preferred enzyme system.

In the context of the foregoing system, the linkers of the inventionwill typically maintain a functional distance between the reactantnucleotide portion and the label fluorophore portion of at least 1 nm,and in preferred aspects, at least 2 nm or greater.

FIG. 3 schematically illustrates a labeled nucleotide analog employingan oligoproline linker, as described previously. As shown, the reactantportion, e.g., nucleotide, is coupled to the label portion, or dye, suchas a fluorophore, via an oligoproline linker that can have a variedlength. While the number of proline monomers may be varied dependingupon the desired application, such linkers will typically be greaterthan 1 nm in length, and thus will typically have at least 3 prolinemonomers (or their equivalents) in the linker (See, e.g., Schuler et al.PNAS 102:2754), e.g., n≧3, e.g., 3, 4, 5, 6, 10 or greater, to yieldlinkers having a functional length that is at least 1 nm or greater andpreferably at least 2 nm or greater.

III. Applications

As noted previously, the compositions described herein are particularlyuseful in real-time analytical reactions where one is observing chemicalreactions through the illumination of the reaction components. In aparticularly preferred aspect, these compositions are useful inreal-time analysis of enzymatic reactions using fluorescent orfluorogenic reactants or products, where such fluorescent or fluorogenicreactants or products may detrimentally impact the enzymes they arereacting with. One particularly important example of such a systemincludes nucleic acid polymerase mediated, template dependent synthesisof nucleic acids, which can be observed using real-time techniques for avariety of desired goals, including in particular, determination ofinformation about the template sequence. A number of methods have beenproposed for determination of sequence information using incorporationof fluorescent or fluorogenic nucleotides into the synthesized strand bya DNA or other polymerase, and the compositions of the invention areapplicable to these methods. While several of these methods employiterative steps of nucleotide introduction, washing, opticalinterrogation, and label removal, preferred uses of these compositionsutilize “real-time” determination of incorporation. Such methods aredescribed in detail in, for example, U.S. Pat. Nos. 7,056,661,7,052,847, 7,033,764 and 7,056,676, the full disclosures of which areincorporated herein by reference in their entirety for all purposes.

Briefly, such methods observe an immobilized polymerase/template/primercomplex as it incorporates labeled nucleotide analogs. Using opticaltechniques that illuminate small volumes around the complex withexcitation radiation, e.g., THU methods, optical confinements like ZeroMode Waveguides (ZMWs) (See, U.S. Pat. Nos. 6,917,726, 7,013,054,7,181,122, 7,292,742 and 7,170,050 and 7,302,146), and the like, one canidentify incorporation events based upon the optical signature of theirassociated fluorophore, as compared to non incorporated, randomlydiffusing labeled nucleotide analogs. By providing each different typeof nucleotide with a distinguishable fluorescent label, e.g., having adistinguishable emission spectrum, one can identify each base as it isincorporated, and consequently read out the sequence of the template asthe nascent strand is created against it. By utilizing the compositionsof the invention, negative impacts of the fluorescent label on thepolymerase or other components of the labeled complex (See, e.g.,published U.S. Patent Application No. 2007/0161017), can be reduced oreliminated by moving the label portion away from the reactant portionand consequently, the active site of the enzyme, or other sensitiveportions of the complex.

IV. Systems

The present invention also employs the nucleotide analog compositionsdescribed herein in conjunction with overall analytical systems.Typically, such systems employ a reaction region that is typicallydisposed in a reaction vessel or well. By way of example, such systemsmay include a substrate component upon which are immobilized, e.g., apolymerase/template/primer complex, for use in the determination ofnucleic acid sequence information of the template, which may be derivedfrom an organism of interest.

Because the compositions of the invention are preferably fluorescentlylabeled, it will be appreciated that the preferred systems of theinvention will comprise fluorescence detection functionalities. Examplesof such systems include those described in, e.g., Published U.S. PatentApplication Nos. 2007/0036511 and 2007/095119 and U.S. patentapplication Ser. No. 11/901,273 filed Sep. 14, 2007, the fulldisclosures of which are incorporated herein by reference in theirentirety for all purposes. One such system is schematically illustratedin FIG. 6.

As shown, the system 600 includes a substrate 602 that includes aplurality of discrete sources of fluorescent signals, e.g., an array ofzero mode waveguides 604. An excitation illumination source, e.g., laser606, is provided in the system and is positioned to direct excitationradiation at the various fluorescent signal sources. This is typicallydone by directing excitation radiation at or through appropriate opticalcomponents, e.g., dichroic 608 and objective lens 610, that direct theexcitation radiation at the substrate 602, and particularly the signalsources 604. Emitted fluorescent signals from the sources 604 are thencollected by the optical components, e.g., objective 610, and passedthrough additional optical elements, e.g., dichroic 608, prism 612 andlens 614, until they are directed to and impinge upon an opticaldetection system, e.g., detector array 616. The signals are thendetected by detector array 616, and the data from that detection istransmitted to an appropriate data processing unit, e.g., computer 618,where the data is subjected to interpretation, analysis, and ultimatelypresented in a user ready format, e.g., on display 620, or printout 622,from printer 624. As will be appreciated, a variety of modifications maybe made to such systems, including, for example, the use of multiplexingcomponents to direct multiple discrete beams at different locations onthe substrate, the use of spatial filter components, such as confocalmasks, to filter out-of focus components, beam shaping elements tomodify the spot configuration incident upon the substrates, and the like(See, e.g., Published U.S. Patent Application Nos. 2007/0036511 and2007/095119 and U.S. patent application Ser. No. 11/901,273, previouslyincorporated herein by reference).

V. Kits

The compositions of the invention are optionally provided in kit form,including various components of an overall analysis in combination withinstructions for carrying out the desired analysis. In particular, suchkits typically include the compositions of the invention, including atleast one, but preferably multiple types of labeled nucleotide analogsof the invention, e.g., A, T, G and C analogs. Each of the differenttypes of labeled nucleotide analogs in the kit will typically comprise adistinguishable labeling group, as set forth above. In addition to theanalog compositions, the kits will optionally include one or morecomponents of a polymerase complex, including, for example polymeraseenzymes, such as any of a number of different types of strand displacingpolymerase enzymes. Examples of such polymerases include, e.g., phi29derived polymerases, and the polymerase enzymes described in, e.g.,Published International Patent Application Nos. WO 2007/075987, WO2007/075873 and WO 2007/076057, the full disclosures of which areincorporated herein by reference in their entirety for all purposes.

Additional reaction components are also optionally included in suchkits, such as buffers, salts, universal priming sequences for initiationof synthesis, and the like. In addition, in particularly preferredaspects, the kits of the invention will typically include a reactionsubstrate that includes reaction regions for carrying out and observingthe synthesis reactions for identification of sequence information. Suchsubstrates include, e.g., multi-well micro or nano plates, as well asarrayed substrates, e.g., planar transparent arrays that includediscrete reaction regions defined by, e.g., structural, chemical orother means. For example, patterned arrays of complexes may be provideddisposed upon planar transparent substrates for observation.Alternatively and preferably, the substrate component comprises an arrayor arrays of optically confined structures like zero mode waveguides.Examples of arrays of zero mode waveguides are described in, e.g., U.S.Pat. No. 7,170,050, the full disclosure of which is incorporated hereinby reference in its entirety for all purposes.

VI. Examples

The use of longer linkers was tested in fluorescently labeled nucleotideanalogs to measure its effect on potential negative impacts ofilluminated analysis on polymerase enzymes incorporating those analogs.In particular, light-induced damage on surface-immobilized DNApolymerase was determined using an assay described as follows.

Biotin-tagged Phi29 DNA polymerase, complexed in a 1:1 stoichiometrywith neutravidin (Pierce), was immobilized on fused silica slides,functionalized with Biotin-polyethylenglycol(PEG)-24-silane by 15′incubation at 0 degrees C. in a buffer containing 50 mM Tris acetate, pH7.5, 75 mM potassium acetate, 0.05% Tween 20 and 5 mM DTT. Unboundpolymerase was washed away using the same buffer, and a 72 bp primed,single-stranded, circular DNA template was bound to the polymerasethereafter in a buffer containing 50 mM ACES, pH 7.1, 75 mM potassiumacetate, 0.05% Tween 20 and 5 mM DTT. Fluorophore-labeled nucleotides(0.25 uM or Alexa 568 dT6P and Alexa 660 dA6P, with and without anaminohexylaminoheptanoic acid linker), unmodified nucleotides (0.25 uM)and manganese acetate (0.7 mM) were added as applicable to initiate DNAsynthesis. The surface-bound polymerase was then exposed to laserillumination by focusing either or both of a 2.1 mW 532 nm laser light,or 2.6 mW of 633 nm laser light (Melles-Griot) an elliptically shapedbeam profile (112 um×5 um at 50% intensity drop, using a cylindricaldefocusing lens at the entrance of an epifluorescence microscope(Olympus)) to the slide surface (exposure step). After laser exposure,the solution was removed and replaced by an identical reaction solutioncontaining unmodified dATP, dCTP, dGTP, dTTP (10 uM) and thebase-labeled nucleotide Alexa Fluor 488-dUTP (1 uM, Invitrogen)(development step). DNA polymerase-mediated incorporation of the latterinto DNA yielded fluorescent DNA strands which were imaged by awide-field epifluorescence microscope, using a Hg arc lamp and standardfilters for Alexa Fluor 488 fluorescence detection.

DNA polymerases that remained active after the laser exposure step inthe presence of phospholinked nucleotide analogs produce a fluorescencesignal while polymerase that were damaged and substantially inactivatedwere incapable of producing fluorescent DNA in the development step. Thesignal levels at the center of the elliptical exposure region werequantitated and normalized using an unexposed surface region of the samechip as a control.

FIG. 5 illustrates a plot of percentage of polymerase activity followingprimer extension with either the Alexa 568 or Alexa 660 labelednucleotide hexaphosphate analogs, and with both a short aminohexyllinker or a longer aminohexyl aminoheptanoic acid linker (“X15”) underlaser illumination by either the 532 nm laser (first bar from left), the633 nm laser (second bar) or an iterative (532+633)(third bar) orconcurrent (532 & 633) combination of the two (fourth bar). As can beseen from the plot, the X15 linker provided enhanced survivability ofpolymerases using both the Alexa568 and Alexa 660 labeled nucleotides inevery case where synthesis was carried out under exposure to at least532 nm wavelength excitation light. The improvements were even moredramatic when the synthesis involved the use of the Alexa660 labelednucleotide under illumination with both the 532 and 633 nm excitationlight.

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. All terms used herein are intended to have theirordinary meaning unless an alternative definition is expressly providedor is clear from the context used therein. To the extent any definitionis expressly stated in a patent or publication that is incorporatedherein by reference, such definition is expressly disclaimed to theextent that it is in conflict with the ordinary meaning of such terms,unless such definition is specifically and expressly incorporatedherein, or it is clear from the context that such definition wasintended herein. Unless otherwise clear from the context or expresslystated, any concentration values provided herein are generally given interms of admixture values or percentages without regard to anyconversion that occurs upon or following addition of the particularcomponent of the mixture. To the extent not already expresslyincorporated herein, all published references and patent documentsreferred to in this disclosure are incorporated herein by reference intheir entirety for all purposes.

What is claimed is:
 1. A labeled nucleotide analog comprising: a labelcomponent, a linker group comprising a double-stranded nucleic acid, anda nucleoside polyphosphate portion, wherein the linker group comprisingthe double-stranded nucleic acid is coupled to the label component andis coupled to the nucleoside polyphosphate portion through a phosphateof the nucleoside polyphosphate portion.
 2. The labeled nucleotideanalog of claim 1 wherein the nucleoside polyphosphate portion has threeto seven phosphates.
 3. The labeled nucleotide analog of claim 1 whereinthe label component comprises a fluorophore.
 4. The labeled nucleotideanalog of claim 1 wherein the label component is coupled to one strandof the double-stranded nucleic acid, and the nucleotide polyphosphateportion is coupled to the other strand of the double-stranded nucleicacid.
 5. The labeled nucleotide analog of claim 1 wherein thedouble-stranded nucleic acid comprise a single molecule withcomplementary portions in which the molecule self-hybridizes to form ahairpin loop structure, and wherein the label component is attached tothe hairpin loop structure.
 6. The labeled nucleotide analog of claim 1wherein the linker group comprises an aliphatic linker group.
 7. Thelabeled nucleotide analog of claim 1 wherein the linker group comprisesthioether linkages, peptide bonds, or maleimide-spacer-NHS linkers. 8.The labeled nucleotide analog of claim 1 wherein the functional distancebetween the nucleoside polyphosphate portion and the label component isat least 1 nm.
 9. The labeled nucleotide analog of claim 1 wherein thefunctional distance between the nucleoside polyphosphate portion and thelabel component is at least 2 nm.
 10. The labeled nucleotide analog ofclaim 1 wherein the double-stranded nucleic acid comprises DNA.
 11. Alabeled nucleotide analog comprising a structure of formula I, II orIII,

wherein, Label is a labeling component, X is a linkage bond, L is alinker group, n is from 3 to 7, and Base is a nucleobase.
 12. Thelabeled nucleotide analog of claim 11 wherein the label componentcomprises a fluorophore.
 13. The labeled nucleotide analog of claim 11wherein the linker group comprises an aliphatic linker group.
 14. Thelabeled nucleotide analog of claim 11 wherein the linker group comprisesthioether linkages, peptide bonds, or maleimide-spacer-NHS linkers. 15.The labeled nucleotide analog of claim 11 wherein the functionaldistance between the nucleoside polyphosphate portion and the labelcomponent is at least 1 nm.
 16. The labeled nucleotide analog of claim11 wherein the functional distance between the nucleoside polyphosphateportion and the label component is at least 2 nm.
 17. A composition forsequencing, comprising a polymerase enzyme and a nucleotide analogsubstrate for the polymerase enzyme, the nucleotide analog substratecomprising: a label component, a linker group comprising adouble-stranded nucleic acid, and a nucleoside polyphosphate portion,wherein the linker group comprising the double-stranded nucleic acid iscoupled to the label component and is coupled to the nucleosidepolyphosphate portion through a phosphate of the nucleosidepolyphosphate portion.
 18. The composition of claim 17 wherein thenucleoside polyphosphate portion has three to seven phosphates.
 19. Thecomposition of claim 17 wherein the label component comprises afluorophore.
 20. The composition of claim 17 wherein the label componentis coupled to one strand of the double-stranded nucleic acid, and thenucleotide polyphosphate portion is coupled to the other strand of thedouble-stranded nucleic acid.
 21. The composition of claim 17 whereinthe double-stranded nucleic acid comprise a single molecule withcomplementary portions in which the molecule self-hybridizes to form ahairpin loop structure, and wherein the label component is attached tothe hairpin loop structure.